System and method of applied radial technology chromatography

ABSTRACT

A system and method of applied radial technology chromatography using a plurality of beads is disclosed, with each bead comprising one or more pores therein having a diameter of about 250 Å to about 5000 Å, and each bead having an average radius between about 100 μm to about 250 μm. Also disclosed are processes for selecting beads for use in a radial flow chromatography column, and for purifying an unclarified feed stream using a radial flow chromatography column.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/595,826, filed on Dec. 7, 2017, the entire contents of which areincorporated by reference herein in entirety.

FIELD OF THE INVENTION

This disclosure relates to radial flow column chromatography, and moreparticularly to a radial flow column comprising beads of specific sizeand parameters to enhance filtration of unclarified feed streams, andmethods of selecting the beads.

BACKGROUND OF THE INVENTION

Chromatography, as it is generally used, is a technique for theseparation of various components of a sample mixture. In a liquidchromatography system, a sample followed by an elution fluid is injectedinto a chromatographic separation column. The separation column containsa packing or matrix medium or material which interacts with the variouscomponents of the sample to be separated. The composition of theseparating medium depends on the fluid being directed therethrough toeffect the desired separation. As the sample and elution fluids passthrough the separating medium, the various components of the sampletravel at different rates through the separating medium as a result ofdifferential interactions. These components emerge separated in theoutlet or effluent from the separation medium.

Various types of the vertical and horizontal flow separation columns areknown in the art. With the need for high performance chromatography,horizontal flow type chromatographic columns were developed. Suchhorizontal or radial flow columns are described in, e.g., U.S. Pat. Nos.4,627,918 and 4,676,898. In the horizontal or radial flow type columns,the sample and elution fluids are introduced via a distributor to theouter periphery or circumferential wall or surface of the separatingmedium or matrix, and the fluids pass horizontally or radially inwardlythrough the separation medium to a central or collection port and thenelute from the column at different times and at different rates.

Later, chromatographic columns and methods were developed for directprocessing of crude feeds for isolation of biologically activematerials, including cell/fermentation harvest, tissue extracts, algae,plant derived cells and materials, and plasma/blood. The large beadchromatography media are packed into a standard, low pressurechromatography column in which end-plate screens are replaced with largepore screens (60-180 μm pores). The large pores prevent column blockage.Because particle sizes are large, the cellular material flows betweenthe beads in the interparticle lumen, while the soluble product iscaptured by functional groups on the beads.

Traditionally, downstream processing of biologics from cellculture/fermentation harvests has required two major operations: i) feedstream preparation and ii) recovery and purification. The sample must beproperly prepared before application to a column. This is both timeconsuming and can be quite costly. If preparation of the sample isneeded, the feed stream is generally diluted to reduce cell density,viscosity, and salt concentration, all of which is beneficial forimproved recovery and purification. Recovery involves the removal ofcellular and other particulate materials by centrifugation and/ormicrofiltration, as well as an initial volume reduction step, typicallyultrafiltration. Since conventional chromatography media are rapidlyfouled by cell debris, particle-free feed must be prepared for thepurification operation.

Centrifugation and filtration are not only lengthy and costlyoperations, they compromise quality. Proteases released from brokencells can degrade the target protein, further complicating the task ofpurification method development and increasing purification costs. Thelonger the contact time with the concentrated cellular debris, the moreproduct may be lost.

Capture of the protein product directly from the unclarified feed wouldminimize product degradation and improve product quality, yield andprocess economy. Also, the capital-intensive recovery operation would begreatly simplified if the product capture and cell removal steps werecombined into a single operation.

There are two approaches to directly capture product from unclarifiedfeed, such as cell culture/fermentation harvest or other biologicalsample (e.g., blood plasma). One approach proposes fluidization of thecapture resin particles. Via fluidization, the individual particles areseparated so that the debris can exit the column bed unobstructed.

This approach suffers from several problems. The fluidized bed systemoperates at a predetermined high flow rate, and there is no flexibilityin the operation or means for changing the size of the column. Thebuffer consumption of the system is higher than on packed bed systems,which is a significant cost factor for high value pharmaceuticalproducts, many of which require specialized buffers for theirpurification. The ratio of column volume to solid phase particle volumeis very high. This will negatively affect residence time and the bindingcapacity within the column, as there is not sufficient time for completediffusion of target molecule into the solid phase. Furthermore,particles will collide within the column bed and solid phase fragmentswill generate so-called “fines”, and reduce both the performance andreuse of the column. Fluidized bed operation also requires specialized,costly hardware and chromatography media.

The other approach is the use of packed bed columns for particulateremoval. This avenue has remained largely unexplored for the followingreason. To clear cellular debris on a packed bed column requires usinglarge, preferably spherical particles. These particles requiresufficient space in the interparticle lumen to let cells or otherparticulates of comparable size exit the column.

The downside of using large particles (beads) is that the proteinbinding capacity is a function of the available surface per unit volumeof gel bed. Therefore with increased particle diameters a loss ofbinding capacity is observed. When the particle diameter is increasedfrom 0.1 mm to 1 mm, such as is required to handle dense cellsuspensions, approximately 90% of the protein binding capacity is lost.This made packed bed columns impractical for processing crude processfeed streams.

Packed bed column operation, however, offers simplicity, efficiency andeconomy. It is flexible and relatively easy to scale. There is no needfor specialized particles, equipment or training of the operators. Theproduction floor-space is relatively small for standard chromatography,and there is no need for the modification of the height of theproduction facility to accommodate the fluidized bed equipment.

Product application rate is another important issue in terms ofthroughput of the operation. This is predetermined for fluidized bedsystems, but for packed column systems just the reaction bindingkinetics is the rate limiting factor. This allows higher throughput, upto 3-10 times higher than for fluidized bed systems.

After product capture, residual cellular material is removed by briefhigh-speed wash pulses. The product is then eluted by typical elutionmethods. Thus, the known large-bead chromatography resins allow directprocessing of cell culture or fermentation broth as well as otherunclarified feeds in a packed bed column by combining cell removal withsimultaneous product capture.

U.S. Pat. No. 5,466,377 proposed a method and large bead chromatographyparticles for the direct capture of a desired product from unclarifiedprocess liquor on standard, low pressure, packed bed chromatographycolumns.

There remains a need for improved chromatographic materials and methodsto achieve direct processing of crude feeds, such as cellculture/fermentation harvests tissue extracts, cell fragments, viruses,blood plasma, waste feed streams derived from vegetable or fruitextracts or waste feed streams derived from milk processing or othernatural material sources, on packed bed columns.

SUMMARY OF THE INVENTION

A radial flow chromatography column is disclosed including: a pluralityof beads, with each bead comprising one or more pores therein, andinterstitial channels formed between the beads. Each pore has a diameterof about 250 Å to about 5000 Å, at least about 80% of the plurality ofbeads have a diameter of about 200 μm to about 500 μm and the beads havean average radius R of between about 100 μm to about 250 μm. The beadsmay be monodisperse (i.e., all beads having a radius of ± about 10% of atargeted or labeled radius) or may have r<0.414 R or r<0.225 R removed.

A process for selecting beads for use in a radial flow chromatographycolumn is also disclosed. That process comprises: a) identifying anarrow desirable bead radius R range based on the components (orparticles of interest) present in the feed stream; b) removing beads ofa defined radius r, which are outside of the desirable bead range; andc) defining the percentage of bead radius R within the desirable beadrange. The beads of radius r may be removed by wet or dry sieving and/orelutriation. The beads being removed may be those having a radiusr<0.414 R or r<0.225 R.

Also, disclosed herein is a process for purifying an unclarified feedstream using a radial flow chromatography column including: a pluralityof beads, with each bead comprising one or more pores therein, andinterstitial channels formed between the beads, wherein each pore has adiameter of about 250 Å to about 5000 Å, at least about 80% of theplurality of beads have a diameter of about 200 μm to about 500 μm andthe beads have an average radius R of between about 100 μm to about 250μm. That process comprises the steps of: a) packing the radial flowcolumn with beads; b) processing a clarified feed stream containing aparticle of interest to calibrate purification conditions; c)determining the binding of the particle of interest from the results ofstep b; and d) processing an unclarified feed stream comprising theparticle of interest.

DETAILED DESCRIPTION

Radial flow columns and methods of making and using the same areprovided for direct filtration, i.e., processing, of crude biologicalfeed streams, e.g., unclarified (i.e., un-filtered) cell cultures. Thesubject disclosure achieves purification at a substantially lower costand with faster processing, especially compared to methods such aspacked bed chromatography and expanded bed chromatography. Exemplaryapplications include fractionation of blood plasma; bioprocessing ofcell cultures to isolate and purify proteins (specificallypharmaceuticals such as herceptin, insulin, avastin, etc.); virus andvirus-like particle capture and purification; purification of waste feedstreams derived from vegetable or fruit extracts, and waste feed streamsderived from milk processing or other natural material sources.

The chromatography disclosed herein surprisingly and beneficially allowswhole cells and other particles to pass undamaged around and between thedisclosed beads without clogging the gel bed, and allows direct passageof unclarified (non-filtered) feed streams of cell cultures, whichcontain whole cells, cell fragments, homogenates of native orrecombinant plant materials, nano-particle solutions, and/or otherparticles (which normally clog columns having small beads). It is alsoable to selectively bind molecular targets of interest in the feedstream, which subsequently may be recovered. For example, for IgGpurification, the beads may be functionized by covalently bindingProtein A or Protein G to the surface of the bead to allow reversibleIgG binding followed by washing and subsequent elution. For virus andVLP capture, the beads may be modified to have a high outer surface areaand high (positive) charge density.

Disclosed herein is a radial flow chromatography column comprising aplurality of beads, with each bead comprising one or more pores therein,and interstitial channels formed between the beads.

The radial flow column may be made in accordance with any known radialflow column, except for the differences discussed herein specificallywith respect to the beads, gel beds, pores and channels. The radial flowcolumn may have a bed length of about 3 cm to about 50 cm and the bedvolume (V) may range from about 5 ml to about 1000 liter. The radialflow column may be shaped as a “donut” (full size radial flow column,having the form of a right circular hollow cylinder), a “cake” slice(having the form of a trapezoidal prism, i.e., small section of donut,having same bed length/radius and curvature but smaller volume), or atruncated “cone” (having the form of a frustum or truncated cone,arising from a small cylindrical core section taken out of cake ordonut, having same bed length/radius and curvature, but even smallervolume than “cake”).

The column may include one or more porous filter frits. Often there isan outer frit and an inner frit. Each frit may be designed to have apore size between about 40 μm to about 300 μm, about 80 μm to about 250μm, or about 100 μm to about 200 μm. The frit may be made of anyconventionally known material. Optionally, it may be made from stainlesssteel, or stainless steel and one or more polymer, such as, but notlimited to polyethylene (PE) or polypropylene (PP).

The radial flow column of the “donut”, “cake” or “cone” shape all haveconstant ratios of the area of the outer frit to the area of the innerfrit. This ratio can be in the range of 1.5:1 to 10:1. The preferablerange is 2:1 to 4:1. The flow dynamics of the three different shapeshaving identical bed lengths and an outer to inner frit area ratio arevirtually identical.

The beads may be spherical or near spherical. The beads may be made of apolymer, glass, alumina, metal or other crystalline, semi-crystalline oramorphous material, silica, controlled pore glass (CPG), cellulose,encapsulated iron particles, encapsulated CPG, encapsulated silica, orany combination thereof. The polymer beads may be made of any polymerknown for use in the art, for example, a polyacrylate, e.g.,methacrylate, a polystyrene, or a polysaccharide, such as dextran,pullulan, agarose, or native or bonded polysilicates. The beads mayconsist of two or more homogeneously or heterogeneously blendedpolymers. The polymer beads may be spherical (or nearly spherical)polysaccharide beads.

The beads may have average diameters of between about 200 μm to about1000 μm, or about 200 μm to about 500 μm. In an embodiment, at leastabout 80% of the polymer beads have a diameter of about 200 μm to about500 μm, or at least about 85%, or at least about 90% of the polymerbeads have a diameter of about 200 μm to about 500 μm.

The beads may have an average radius (R) of between about 100 μm toabout 500 μm, or about 100 μm to about 250 μm.

Most beads are not generally monodisperse (all the same size with thesame diameter) but have a range of diameters which extend out beyond thegiven range. Therefore, for example this measurement means that 80% ofthe total mass (or volume) of the beads falls within 200-500 μm. Theother 20% (independently of how the percentage is defined) is outsidethe range, either smaller or larger. In order for the interstitialchannels to not clog, it is important that the smaller beads in the 20%outside the given range are removed or at least depleted at some timeprior to packing the column. If the smaller beads that are nearly thesame size as the diameter of the channel are not depleted or removed, agel bed of beads between 200-500 μm may contain enough small beads topartially or completely block the channels.

Some chromatography beads are commercially available and often are soldby bead diameter. However, the given diameter is an average of beaddiameters; it does not mean that all of the beads have that givendiameter. Other companies may sell beads by listing a range of beaddiameters. However, this is the range in which a certain, sometimesundefined, percentage of beads falls within. The total percentage ofbeads having a diameter which falls outside the given range is thenunknown; there is rarely a given percentage of beads which fall above orbelow the range.

The beads may have functionalized groups (e.g., ionic exchange groups,hydrophobic interaction groups, etc.), allowing them to selectively bindmolecular targets of interest (for potential later recovery). Potentialtargets include viruses, virus-like particles, proteins (specifically,but not limited to, IgG, IgM, IgY, and blood proteins), DNA, RNA,oligonucleotides, polypeptides and cells.

The beads have one or more pores therein. Each pore has a diameter ofabout 250 Å to about 5000 Å. Each pore may extend partially through thebead resulting in a dead-end, or may go all the way through the bead toanother exit point.

Interstitial channels are formed between packed beads and partiallycomprising the void volume. When these channels are wide enough to allowthe cells and cell fragments to pass through the gel bed withoutclogging and when the channels are free of smaller beads which, due totheir size, could restrict or block the passage of cells and cellfragment, the user can utilize the packed beads to avoid deleteriousfiltration or centrifugation, precipitation or other costly,time-consuming and potentially product-losing steps prior to thechromatography purification step.

Monodisperse spherical beads will ideally pack in either hexagonal closepacking (HCP) or cubic close packing (CCP) arrangements. Both packingarrangements have the same maximum bead (sphere) density and both havesimilar packing energies, so neither is clearly energetically favoredover the other. Although the preferred packing arrangement of polymer,e.g., polysaccharide, beads is not predictable, both packingarrangements form channels (interstitial space or channels) between thebeads, the smallest size of which is important to the success or failureof the device and system disclosed herein.

The interstitial channels should be: 1) large enough to allow cells,cell fragments and other interfering particles to pass through withoutclogging the gel bed; 2) free of smaller beads which are of similar sizeto the interstitial channel itself and could therefore clog thechannels; and 3) formed from a population of beads having as narrow arange of bead diameters as possible, with monodisperse beads being asclose to ideal size as possible.

In addition, the interstitial channels should not be compressed toonarrowly, which could clog the interstitial channels, by creating: i)too high a flow rate, ii) too high pressure, or iii) too high denselypacking of the beads. The interstitial channels should be open (i.e.,free from clogs) to provide a continuous path for processing the feedstream. Also, too high of a flow rate will narrow the interstitialchannels of a non-rigid gel bed. Therefore, the gel bed must have enoughrigidity to allow flow rates of 0.1 column volumes to 10 column volumesper minute without narrowing the interstitial channels (through beadcompression) to allow cells and cell fragments to pass through the gelbed.

Rigidity of the gel bed also depends on the rigidity of the beadsthemselves. To minimize changes in the diameter/size of the interstitialchannels between beads (assuming all beads are spherical ornear-spherical and with a given size distribution), it is important thatthe morphology of the beads does not change under flow conditions.

One option is to use very rigid beads that are inert to the buffers usedin chromatographic separations and thus will exhibit no change in theirsize or shape; however, beads made from silica or CPG (Controlled PoreGlass), for example, which have excellent mechanical rigidity andstability, have little or no tolerance towards NaOH.

Polymer beads, such as, but not limited to methacrylate or polystyrenebeads, can be made more reagent stable and retain rigidity.Polysaccharide beads, such as, but not limited to dextran or agarosebeads, are less rigid but are more stable to reagents such as NaOH.However, polysaccharide beads are often “softer” than other polymerbeads and thus more prone to becoming compressed. The degree to whichcompression occurs will depend on the applied pressure from liquidflowing through the packed gel bed. Too much compression will result ina non-porous bed through which no flow is possible (closure of bothinternal pore network and interstitial spaces). The key factors whichusually lead to this increase in pressure and compression are the flowrate of the applied liquid (normally expressed as mL/min, CV/min orcm/hr) and the viscosity of the liquid.

The mechanical stability and rigidity of softer polysaccharide beads maybe enhanced by applying certain procedures. For example, the followingapproaches will improve mechanical stability and rigidity of the beads:

-   -   1. Crosslinking within the polysaccharide structure. This will        make the beads more resistant to pressure and thus preserve the        bead morphology. However, depending on the crosslinking        chemistry applied, the size of the internal pores within the        bead structure may be affected.    -   2. Increasing the density of the amount of polysaccharide used        in the formulation of the beads. This is often done with        agarose-based particles. Standard commercially available agarose        beads have agarose percentages between about 2 and 10% (20 to        100 grams of agarose per litre of formulated beads). A bead        having increased density may have ≥ about 6% agarose. However,        this increase in density may reduce the internal pore sizes of        the beads:        -   4% beads have an average molecular weight cut-off of around            20 million Dalton        -   6% beads have an average molecular weight cut-off of around            4 million Dalton

The optimal interstitial channel size, which is dependent on the beadsize, ultimately depends upon the components present in the feed stream.Specifically, the optimal size of the interstitial channels depends onthe particles that are to desirably pass through the gel bed. Theprocess of determining the best size for the interstitial channels maybe as follows:

-   -   1. Determination of Bead Size: From the radius of the cells or        fragments that are to pass through the gel bed, estimate the        required radius of the channel formed by three beads (the        “Narrowest Channel Radius”) and calculate the minimum bead size        necessary.    -   2. Determination of Size Fraction to be Removed from Gel: From        the radius of the beads (monodisperse bead radius or average of        bead radii for polydisperse beads), calculate the length of the        radii of the tetrahedral and octahedral sites. The radii of        these sites are those of the largest beads which must be removed        from the gel.

Then the theoretically largest diameter of a small sphere which can fitthrough the smallest channel formed between three perfect identicalspheres has been calculated. The diameter of the channel may be about 3to about 10-fold larger, or about 4 to about 6-fold larger than thelargest particle (i.e., cell, cell fragment, or other particle) that ispresent in the unclarified feed stream.

For Cubic Close, Hexagonal Close, or Barlow Packing of monodispersebeads, fully settled, vibrated to no measurable change in the packingdensity (Kepler Conjecture), the following holds true:

-   -   Tetrahedron site radius r_(tet)=0.225 R (R is the Radius of the        bead, r_(tet) is the radius of the Tet-site)    -   Octahedron site radius r_(oct)=0.414 R (R is the Radius of the        bead, r_(oct) is the radius of the Oct-site)    -   Narrowest Channel radius r_(cha)=0.155 R (R is the Radius of the        bead, r_(cha) is the radius of the channel)    -   Beads having a radius of x, where 0.155 R<x<0.414 R have a size        which can fit into and permanently occupy an Octahedron site.    -   Beads having a radius of x, where 0.155 R<x<0.225 R have a size        which can fit into and permanently occupy a Tetrahedron site.

Both tetrahedron and octahedron “holes” are always present in a bed ofpacked beads. The holes are named tetrahedron or octahedron based on thenumber of beads that surround and form the hole.

For Random Close Packing of polydisperse beads, the above values are aminimum and actual values may be larger. Channels of radiusr_(cha)=0.155 R should be a minimum of 1.1-fold (preferably about 2 toabout 4-fold) the radius of the cell, fragment or particulate to passeasily through the gel bed.

The monodisperse beads may be prepared by:

-   -   a. manufacturing monodisperse beads with an optimal radius and        complete absence of smaller beads;    -   b. manufacturing a narrow range of polydisperse beads by        carefully controlling the conditions during an emulsion process.        These conditions include: addition of an optimal type and amount        of emulsifier, maintaining an optimal stirring speed,        maintaining an optimal temperature, all which contribute to a        narrowing of the size distribution of beads formed;    -   c. wet or dry sieving to remove the fraction of particles        smaller (or larger) than a selected size; and/or    -   d. elutriation to remove the fraction of particles smaller than        a determined size.

To improve filtration, smaller beads that could clog the interstitialchannels may be removed prior to packing of the gel bed.

The interstitial channels may be improved in form and function by takingsome or all of the following steps:

-   -   a. Calculate/determine the desirable average bead radius R to        allow cells, fragments of cells, or other particulates to pass        through, for example, using the information shown in Tables 1-3        below.    -   b. Form a gel bed where beads having radius r<0.414 R have been        removed.        -   i. Removal of all beads having radius r<0.414 R will create            a gel bed with maximum flow and porosity, and shorter path.            A benefit thereof is faster purification processing.    -   c. Form a gel bed where beads having radius r<0.225 R have been        removed.        -   i. Removal of all beads having radius r<0.225 R will create            a gel bed with somewhat decreased porosity, an increased            path length, and increased residence time. A benefit thereof            is more efficient purification.    -   d. Removal of the smaller beads having radius r<0.225 R or        r<0.414 R will prevent blockage/clogging of interstitial        channels in the gel bed. This will also reduce the amount of        cleaning of gel bed required between purification cycles.    -   e. Increase the radius r of the beads to be removed by up to 25%        (i.e., the beads being removed have a radius r that is 25%        larger than otherwise indicated).    -   f. Narrow the bead size distribution as much as possible to        achieve reduced Random Close Packing density and to approach        Cubic Close/Hexagonal Close packing density.

TABLE 1 Average Bead Interstitial Channel diameter (μm) diameter (μm)100 15.5 150 23.2 200 30.9 Target 250 38.7 Channel 300 46.4 Sizes 35054.1 400 61.9 450 69.6 500 77.4 750 116 1000 154.7

TABLE 2 Will fit into Cell/ Average Interstitial Particle of DiameterChannels of interest (μm) (μm, 3-fold max) E. Coli Cell 2 6 HEK-293 Cell13 39 CHO Cell 15 45 Yeast Cell 5 15 Tobacco BY-2 35-100 105-300 CellVero Cell 8 24 Virus 0.017-0.5    0.05-1.5  Plasmid DNA 0.001-0.003 0.003-0.010

TABLE 3 Protein/ Average Will fit Particle Diameter into Bead Pores ofinterest (Å) of (Å, 6-fold max) IgG 120 720 IgM 350-600 2100-3600 Plasmid DNA 12-30 72 Virus  170-2500 1020-15000

Another embodiment is a process for selecting beads for use in a radialchromatography gel bed which includes: a) identifying a narrow desirablebead diameter (or radii) range based on the components (or particles ofinterest) present in the feed stream; b) removing beads of a defineddiameter (or radii) outside of the desirable bead diameter range; and c)defining the percentage of bead diameters (radii) within the desirablebead diameter range.

Yet another embodiment is a process for filtering an unclarified feedstream using a radial flow column disclosed above comprising the stepsof:

-   -   a. packing the radial flow column with beads;    -   b. processing a clarified feed stream containing a particle of        interest to calibrate purification conditions;    -   c. determining the binding of the particle of interest from the        results of step b; and    -   d. processing an unclarified feed stream comprising the select        protein.

The particle of interest may be a whole cell, cell fragment, virus, orprotein. It may be a VLP, DNA, RNA, antigen, liposome, oligo- orpolysaccharide.

After packing the radial flow column with the beads, a clarified feedstream is used for calibrating optimal purification conditions prior toactual routine purification of unclarified feed streams. This providesan understanding of how the particle of interest, such as protein, bindson the column (RFC, Zetacell™). For purification of unclarified streams,both forward and backward washing may be used to remove cell/cellfragment traces. Cake or cone shapes may be used for small scaleoptimization before scaling up/using donut shape. The process may beused in conjunction with “simulated moving bed” (“SMB”)/continuousprocesses.

Depending on the reagent systems used during chromatography, the polymerbeads will shrink and swell, thereby increasing and decreasing bothinternal pore diameters as well as the interstitial spaces between the(spherical) beads. This does not occur with silica and CPG particles,but occurs for all polymer and polysaccharide particle-based gel beds.The effects of the swelling and shrinking may be controlled, inentirety, in near entirety, or at least in part, by the ordinarilyskilled artisan's careful selection of the buffer system used to packthe column and used during routine operation.

After optimizing the process with clarified feed streams directly on asmall radial flow chromatography column containing polymer beads as agel bed, linear scale up to a larger process scale may be achieved bymaintaining the bed length, maintaining the ratio of outer to inner fritareas and maintaining all operational parameters (flow rate, number ofcolumn volumes per unit time, buffer composition, residence time, solidphase, pressure, and temperature) for each step of the process (loadingof clarified or unclarified feed stream, washing in forward and reversedirections to remove cells, cell debris and non-bound materials, elutionto release directly captured target from the solid phase, regenerationto clean and prepare the column and solid phase for subsequent reuse).If at any time there is a need to re-calibrate, re-optimize or otherwisechange the large scale purification system, linear scale down to amanageable and small RFC column is also achieved by maintaining the bedlength, maintaining the ratio of outer to inner frit areas andmaintaining all operational parameters (flow rate, number of columnvolumes per unit time, buffer composition, residence time, solid phase,pressure, and temperature) for each step of the process (loading ofclarified or unclarified feed stream, washing in forward and reversedirections to remove cells, cell debris and non-bound materials, elutionto release directly captured target from the solid phase, regenerationto clean and prepare the column and solid phase for subsequent reuse).

The foregoing illustrates some of the possibilities for practicing theinvention. Therefore, although specific example embodiments have beendescribed, it will be evident that various modifications and changes maybe made to these embodiments without departing from the broader scope ofthe invention; many other embodiments are possible within the scope andspirit of the invention. Various features are grouped together in asingle embodiment for the purpose of streamlining the disclosure. Thismethod of disclosure is not to be interpreted as reflecting that theclaimed embodiments have more features than are expressly recited ineach claim. Rather, as the following claims reflect, inventive subjectmatter lies in less than all features of a single disclosed embodiment.Thus the following claims are hereby incorporated into the aboveDescription of the invention, with each claim standing on its own as aseparate example embodiment.

It should be noted that it is envisioned that any feature or elementthat is positively identified in this document may also be specificallyexcluded as a feature or element of an embodiment of the presentinvention as defined in the claims. It should also be noted that it isenvisioned that any feature or element that is positively identified (orthat is excluded, either specifically or by implication) may be used incombination with any other feature or element that is positivelyidentified (or that is excluded, either specifically or by implication).

What is claimed:
 1. A radial flow chromatography column systemcomprising: a radial flow chromatography column having a bed length ofabout 3 cm to about 50 cm, a plurality of beads packed in the radialflow chromatography column, and unclogged interstitial channels formedbetween the beads, wherein each bead comprises one or more pores, andeach pore has a diameter of about 250 Å to about 5000 Å, wherein atleast about 80% of the plurality of beads have a diameter of about 200μm to about 500 μm, wherein the beads have an average radius R of about100 μm to about 250 μm, and wherein the beads maintain their size andmorphology under flow rates to 10 column volumes per minute, wherein thebeads are monodisperse wherein all beads have a radius of ± about 10% ofa targeted or labeled radius and smaller beads having a radius r<0.225 Rhave been removed prior to packing the column, and wherein the systemprovides for purifying a particle of interest by direct filtration froman unclarified feed stream or crude biological feed stream.
 2. Theradial flow chromatography column system of claim 1, wherein the bead ismade of a polymer, glass, alumina, silica, controlled pore glass (CPG),cellulose, encapsulated iron particles, encapsulated CPG, orencapsulated silica.
 3. The radial flow chromatography column system ofclaim 2, wherein the bead is a polymer bead.
 4. The radial flowchromatography column system of claim 1, wherein any beads havingr<0.414 R have been removed.
 5. The radial flow chromatography columnsystem of claim 1, wherein the interstitial channels have: a tetrahedronsite radius r_(tet)=0.225 R, an octahedron site radius r_(oct)=0.414 R,or both.
 6. The radial flow chromatography column system of claim 1,wherein the interstitial channels have a narrowest channel radiusr_(cha)=0.155 R.
 7. The radial flow chromatography column system ofclaim 1, wherein the particle of interest is a protein, virus, VLP, DNA,RNA, antigen, liposome, oligo- or polysaccharide, or any combinationthereof.
 8. A process for selecting beads for use in the radial flowchromatography column of claim 1 comprising: a) identifying a narrowdesirable bead radius R range based on the components present in thefeed stream; b) removing beads of a defined radius r, which are outsideof the desirable bead range; and c) defining the percentage of beadradius R within the desirable bead range.
 9. The process of claim 8,wherein the bead is made of a polymer, glass, alumina, metal or othercrystalline, semi-crystalline or amorphous material, silica, controlledpore glass (CPG), cellulose, encapsulated iron particles, encapsulatedCPG or encapsulated silica.
 10. The process of claim 8, wherein the beadis a polymer bead.
 11. The process of claim 8, wherein the beads ofradius r are removed by wet or dry sieving, and/or elutriation.
 12. Theprocess of claim 8, wherein beads having radius r<0.414 R are removed.13. A process for purifying an unclarified feed stream using the radialflow chromatography column of claim 1 comprising the steps of: a.packing the radial flow column with the beads; b. processing a clarifiedfeed stream containing a particle of interest to calibrate purificationconditions; c. determining the binding of the particle of interest fromthe results of step b; d. processing an unclarified feed streamcomprising the particle of interest.
 14. The process of claim 13,wherein the particle of interest is a protein, virus, VLP, DNA, RNA,antigen, liposome, oligo- or polysaccharide, or any combination thereof.15. A radial flow chromatography filtration system for isolating one ormore particle of interest comprising: a radial flow chromatographycolumn having a bed length of about 3 cm to about 50 cm, a plurality ofbeads packed in the radial flow chromatography column, uncloggedinterstitial channels formed between the beads, and an unclarified feedstream comprising a plurality of particles; wherein each bead comprisesone or more pores therein, and each pore has a diameter of about 250 Åto about 5000 Å, wherein at least about 80% of the plurality of beadshave a diameter of about 200 μm to about 500 μm, wherein the beads havean average radius R of about 100 μm to about 250 μm, wherein the beadsmaintain their size and morphology under flow rates to 10 column volumesper minute, wherein the beads are monodisperse wherein all beads have aradius of ± about 10% of a targeted or labeled radius and smaller beadshaving a radius r<0.225 R have been removed prior to packing the column,and wherein the diameter of each of the interstitial channels is about 3to about 10-fold larger than the largest particle in the unclarifiedfeed stream.
 16. The radial flow chromatography filtration system ofclaim 15, wherein the bead is made of a polymer, glass, alumina, silica,controlled pore glass (CPG), cellulose, encapsulated iron particles,encapsulated CPG, or encapsulated silica.
 17. The radial flowchromatography filtration system of claim 16, wherein the bead is apolymer bead.
 18. The radial flow chromatography filtration system ofclaim 15, wherein the particle of interest is a protein, virus, VLP,DNA, RNA, antigen, liposome, oligo- or polysaccharide, or anycombination thereof.
 19. A process for selecting beads for use in theradial flow chromatography column of claim 15 comprising: a) identifyinga narrow desirable bead radius R range based on the components presentin the feed stream; b) removing beads of a defined radius r, which areoutside of the desirable bead range; and c) defining the percentage ofbead radius R within the desirable bead range.
 20. The process of claim19, wherein the bead is made of a polymer, glass, alumina, metal orother crystalline, semi-crystalline or amorphous material, silica,controlled pore glass (CPG), cellulose, encapsulated iron particles,encapsulated CPG or encapsulated silica.
 21. The process of claim 19,wherein the bead is a polymer bead.
 22. The process of claim 19, whereinthe beads of radius r are removed by wet or dry sieving, and/orelutriation.
 23. The process of claim 19, wherein beads having radiusr<0.414 R are removed.